Leonardo D'Aloiso

Novel somatic MEN1 gene alterations in sporadic primary hyperparathyroidism and correlation with clinical characteristics

Primary hyperparathyroidism (pHPT) is a common endocrine disease that in more than 95% of cases is sporadic and only in some cases is caused by inherited disorders, isolated or as part of multiple endocrine neoplasia (MEN1 and 2). Somatic mutations of MEN1 gene have also been described in sporadic parathyroid tumors. In our study, we examined the presence of alterations in MEN1 gene in a series of 39 patients who had undergone surgery for sporadic pHPT (35 with parathyroid adenoma or hyperplasia, 4 with a carcinoma). A genotype-phenotype correlation was also analysed. After DNA extraction from paraffin-embedded tissues, we amplified by PCR and sequenced the exons 2–10 of the MEN1 gene. Somatic MEN1 mutations were detected in 6 of the 35 patients with a benign parathyroid lesion examined (17.1%), whereas no alterations were found in the carcinomas. Four novel MEN1 gene mutations were identified as follows: one frameshift mutation (222insT, exon 2), one frameshift deletion (912delTA, exon 5), one in-frame deletion (835del18, exon 4) and one missense mutation (P291A, exon 6). In addition, one missense mutation (L89R, exon 2) and one nonsense mutation (Q536X, exon 10) were previously reported. Moreover, two polymorphisms were also found: one allele carried a R171Q polymorphism (1/39 tumors), while a D418D polymorphism (GAC/GAT) was found in 15 and 8 tumors in hetero (CT) and homozygosity (TT), respectively. In no case (mutations and/or polymorphisms) did we find a genotype-phenotype correlation. In conclusion, our data demonstrate the presence of somatic alterations of the MEN1 tumor suppressor gene in about one fifth of benign sporadic parathyroid tumors. The absence of a genotype-phenotype correlation, however, suggests the involvement of other genetic/epigenetic factors for the full expression of the disease.

Global skeletal uptake of 99mTc-methylene diphosphonate (GSU) in patients affected by endocrine diseases: Comparison with biochemical markers of bone turnover

Abstract: This study aimed to clinically validate the global skeletal uptake (GSU) of 99mTc-methylene diphosphonate (99mTc-MDP), and to compare it with a marker of bone formation (i.e. serum osteocalcin or OC) and an index of bone resorption (i.e. urinary deoxypyridinoline or U-DPD) in different endocrine disorders affecting the skeleton. We studied 29 female patients with thyrotoxicosis (TT), 27 with primary hyperparathyroidism (PHPT), 16 with acromegaly (AC), 15 with Cushing’s syndrome (CS), and altogether 110 healthy women matched for age, BMI and menstrual status. In all subjects total body digital scan images (TBDS) were acquired at 5 min and at 4 h after the administration of 99mTc-MDP; the whole body retention (WBR) of the tracer was measured by counting two identical sets of rectangular ROIs, and GSU was subsequently calculated by drawing an irregular ROI on 4 h TBDS images. Serum OC was assessed by IRMA and urinary DPD by fluorometric detection after reverse phase high pressure chromatography. In TT patients GSU (40.0 ± 5.1 vs 36.5 ± 4.8%), OC (19.1 ± 11.8 vs 7.1 ± 2.9 mg/l) and U-DPD (62.4 ± 42.7 vs 19.5 ± 5.3 pmol/pmol) were significantly (p<0.01) higher than in controls. PHPT patients showed GSU (47.2 ± 6.6 vs 37.8 ± 5.3%), OC (38.6 ± 40.9 vs 8.2 ± 2.5 mg/l), and U-DPD (55.0 ± 51.3 vs 21.9 ± 6.1 pmol/pmol) values significantly (p<0.001) higher than controls. In CS patients, GSU (39.6 ± 6.4 vs 32.7 ± 3.5%; p<0.01) and U-DPD (22.8 ± 8.4 vs 16.5 ± 2.7 pmol/pmol; p<0.05) were higher, whereas OC (3.6 ± 2.4 vs 5.2 ± 1.9 mg/l; p<0,05) was lower than in controls. In AC patients, GSU (34.9 ± 5.3 vs 35.2 ± 3.4%) did not differ significantly from controls, whereas OC (16.8 ± 8.8 vs 6.9 ± 2.9 mg/l; p<0.001) and U-DPD (30.9 ± 13.6 vs 21.0 ± 5.7 pmol/pmol; p<0.01) were higher. Stepwise multivariate linear regression analysis was performed with disease activity, creatinine clearance, age, and years since menopause as predictor variables and GSU or OC or U-DPD as dependent variables. The significant partial regression coefficients (r) were: in TT, free triiodothyronine (fT3) with GSU (r = 0.37; p<0.005), Ln OC (r = 0.30; p = NS), Ln U-DPD (r = 0.76; p<0.0001), respectively; in PHPT, PTH with GSU (r = 0.74; p<0.001), Ln OC (r = 0.50; p<0.05), Ln U-DPD (r = 0.64; p<0.001); in CS Ln urinary free cortisol with OC (r = −0.68; p<0.001) and U-DPD (r = 0.66; p<0.05). Our data suggest that GSU could represent a valuable clinical tool for evaluating bone turnover rate in PHPT, CS, TT but not in AC. The behavior of GSU and OC and U-DPD is non-uniform in disorders characterized by a marked uncoupling between bone formation and resorption.

Evaluation of a DHPLC-based assay for rapid detection of RET germline mutations in Italian patients with medullary thyroid carcinoma.

Causative gain-of-function mutations of the RET tyrosine-kinase receptor gene have been reported in more than 95% of inherited cases of medullary thyroid carcinoma (MTC; OMIM# 155240). Most RET activating mutations are clustered in mutational “hot spots” in exons 10, 11, 13, 14, 15 and 16 and are usually detected by single- strand conformation polymorphism (SSCP) followed by direct sequencing. To improve sensitivity, time and costs of mutational screening we have developed a denaturing high performance chromatography (DHPLC) protocol, based on the detection of heteroduplex molecules by ion-pair reverse-phase liquid chromatography under partially denaturing conditions. The mutational screening of RET exons 10, 11, 13–16 was performed in a total of 111 subjects, including 45 MTC patients and 49 relatives with known RET mutations and 17 individuals, being at risk of hereditary MTC and carrying unknown RET alleles. Heteroduplex peaks with a distinct and reproducible DHPLC elution profile allowed the detection of both rare and common RET mutations. Overall, the DHPLC-based methodology showed a high level of sensitivity and accuracy, nearing 100%. Furthermore, our protocol showed the ability to identify: 1) all the mutated codons of RET located in the “hot spots” domain; 2) the different point mutations occurring in the same codon of RET gene; 3) less frequent or rare mutations; 4) polymorphisms. As such, it can be proposed as a relatively simple and highly accurate method for a rapid genetic testing for members of MTC families.